608

Matricellular proteins: extracellular modulators of cell function

Paul Bornstein* and E Helene Sage
The term matricellular has been applied to a group of
extracellular proteins that do not contribute directly to the
formation of structural elements in vertebrates but serve to
modulate cellmatrix interactions and cell function. Our
understanding of the mode of action of matricellular proteins
has been advanced considerably by the recent elucidation of
the phenotypes of mice that are deficient in these proteins. In
many cases, aspects of these phenotypes have illuminated
previously unsuspected consequences of the lack of
appropriate interactions of cells with their environment.
Addresses
*Departments of Biochemistry and Medicine, Box 357350,
University of Washington, Seattle, WA 98195, USA;
e-mail: bornsten@u.washington.edu
Division of Vascular Biology, The Hope Heart Institute,
1124 Columbia Street, Suite 720, Seattle, WA 98104, USA;
e-mail: hsage@hopeheart.org
Current Opinion in Cell Biology 2002, 14:608616
0955-0674/02/$ see front matter
2002 Elsevier Science Ltd. All rights reserved.
Published online 5 August 2002
Abbreviations
CAM
chorioallantoic membrane
CCN
cyr-61, CTGF, Nov
cdk
cyclin-dependent kinase
CTGF
connective tissue growth factor
EC
endothelial cell
ECM
extracellular matrix
MAPK mitogen-activated protein kinase
MMP
matrix metalloproteinase
OPN
osteopontin
PDGF platelet-derived growth factor
SMC
smooth muscle cell
SPARC secreted protein, acidic and rich in cysteine
transforming growth factor
TGF-
TSP
thrombospondin
VEGF
vascular endothelial growth factor

Introduction
In vertebrates, the extracellular matrix (ECM) determines
the physical properties of tissues, as well as many of the
characteristics of the cells within them. In some cases, the
same constituent matrix protein can perform these different functions. Thus, collagen-I, the prototypic structural
protein in bone, tendon and ligament, also serves as an
adhesive protein, and monomeric and polymeric forms of
the protein affect cells differently [1]. It is nevertheless
useful for heuristic purposes to identify different functional
compartments within the ECM, and to categorize the
proteins within them.
The pericellular matrix refers to a subcompartment of the
ECM, adjacent to the cell, which contains growth factors,
cytokines, proteases, and other bioactive molecules that
interact with the cell surface. These proteins, together

with proteoglycans, polymeric collagen and fibronectin

fibrils, and a variety of other soluble proteins, influence
many properties of cells, including their motility, proliferation,
state of differentiation, and predisposition to apoptosis. In
some cells, specialized forms of the pericellular matrix,
termed basement membranes or basal laminae, subserve
a physical role, but these structures also influence cell
function and fate.
During the past 1015 years, increasing attention has been
paid to a group of matrix proteins that modulate cell function
but do not appear to contribute directly to the organization
or physical properties of structures such as fibrils or basal
laminae. These proteins have been termed matricellular
proteins to emphasize their roles as regulators of cell function [2,3]. To the original group of matricellular proteins,
thrombospondin-1 (TSP1), SPARC (secreted protein,
acidic and rich in cysteine; also known as osteonectin), and
tenascin-C [4], can now be added TSP2, osteopontin
(OPN), and possibly the CCN (cyr-61, CTGF [connective
tissue growth factor], Nov) family of proteins [5] and
tenascin X [6]. The properties of matricellular proteins
that justify their inclusion in a separate functional category
of ECM proteins are summarized in Table 1. However,
given the ability of proteins to perform both structural and
cell-regulatory roles, and the ingenuity of nature in recruiting
proteins to perform functions that surprise us, such groupings
should remain fluid. Much of the justification for distinguishing matricellular from matrix proteins has come from
an appreciation of the unusual phenotypes of mice that
lack a matricellular protein. These phenotypes (Table 2)
are superficially mild and are consistent with a minimal
contribution of the proteins to structural integrity. Several
recent comprehensive reviews of individual members of
the matricellular protein family have been published
[5,711,12,13,14]. In this review, we focus on the TSPs
and SPARC; but we discuss recent work on the other
proteins, in an attempt to deduce common mechanisms in
the action of matricellular proteins.

Cell adhesion, migration and chemotaxis

In contrast to matrix proteins that generally foster strong
cell adhesion, TSP1 and 2, tenascin-C and SPARC support
a state of intermediate adhesion, characterized by disruption
of focal adhesions and a reorganization of actin stress fibers
[4,15]. Recent experiments have demonstrated an
interaction between the amino-terminal heparin-binding
domain of TSP1 and cell-surface calreticulin, and suggest
that the resulting signals mediate disassembly of focal
adhesions [16]. An analogous receptor that mediates the
interaction of SPARC with the cell surface and promotes
focal adhesion disassembly has not been identified,
although the activity of SPARC is sensitive to inhibitors of
tyrosine kinase phosphorylation [8]. X-ray crystallography

Matricellular proteins: extracellular modulators of cell function Bornstein and Sage

studies reinforce the idea that SPARC is poised to act

between the cell surface and the ECM, as residues identified
for cell binding, inhibition of adhesion, and diminution of
focal adhesions cluster on one face of the protein, whereas
collagen-binding regions lie opposite [17]. Evidence now
favors SPARC as an antagonist of integrinECM interactions,
a mechanism by which its potent and widespread de-adhesive
effects could occur. By analogy, tenascin-C, which disrupts
the adhesion of cells to fibronectin, interacts with the
HepII site of fibronectin and prevents its binding to the
signaling cell-surface receptor, syndecan 4 [18]; previously,
the interaction of an alternatively spliced form of tenascin-C
with annexin II had been implicated in this activity [15].
In apparent contrast to most of the other matricellular
proteins, OPN facilitates the adhesion of different cell
types to the ECM via its interaction with several integrins,
although its stimulatory effects on, for example, macrophage
activation and migration indicate that the state of adhesion
might be intermediate [10]. It should be noted that studies
of cell adhesion and migration performed on planar substrates
in vitro should take into account important differences in
cell behavior in vivo. Thus, a three-dimensional environment has been shown recently to increase the rate of
adhesion, the molecular composition of focal and fibrillar
adhesions, and other properties of fibroblasts [19].
The disruption of the Thbs2 gene in mice has indirectly
provided a wealth of information regarding the function of
TSP2 (Table 2). In an attempt to discern a cause for abnormal
collagen fibrillogenesis, Yang et al. [20] cultured dermal
fibroblasts from TSP2-null mice and detected a substantial
reduction in both adhesion and spreading on a variety of
substrates, including TSP2. This finding was unexpected,
since TSP2, by analogy with TSP1, was predicted to exert
a de-adhesive action on cells. Normal adhesion was
restored by introduction of a TSP2 cDNA or by prolonged
incubation in the presence of soluble recombinant TSP2.
The adhesive defect was shown to result from an increase
in matrix metalloproteinase (MMP)-2 in TSP2-null cells
and culture medium [20]. It is now established that both
TSP1 and TSP2 bind MMP2 and that the TSP2MMP2
complex is endocytosed by the low-density lipoprotein
receptor-related protein (LRP) [21]. Thus, TSP2 serves to
clear MMP2 from the pericellular environment. Although
normal dermal fibroblasts also secrete TSP1 in vitro, neither
TSP1- nor TSP2-null cells compensate by increased
production of the paralogous protein [22].
In TSP2-null mice, MMP2 levels are increased in the foreign
body response to subcutaneously implanted sponges [23]
and in healing excisional wounds (A Agah, personal communication). MMP2 is therefore likely to contribute to the
increased angiogenesis that is observed in these responses
to injury, as it does in other instances of angiogenesis [24].
Increased MMP2 levels can also stimulate the migratory
activity of fibroblasts during the proliferative phase of
wound healing, and could account, together with increased

609

Table 1
Some distinguishing characteristics of matricellular
proteins.
Expressed at high levels during development and in response to
injury
Do not subserve structural roles but function contextually as
modulators of cellmatrix interactions
Bind to many cell-surface receptors, the ECM, growth factors,
cytokines and proteases
Generally induce de-adhesion, in contrast to the adhesivity of most
matrix proteins
In most cases, targeted gene disruption in mice produces either a
grossly normal or a subtle phenotype that is exacerbated upon
injury

angiogenesis, for the accelerated healing seen in TSP2-null

mice [7]. Thus, by modulating the levels of a key protease
in matrix turnover, TSP2 amply fulfils its role as a matricellular protein, although it is still not clear whether the
targets of MMP2 action are predominantly matrix proteins,
or include cell-surface receptors involved in cell adhesion
and migration.
To determine whether or not the effects of a lack of TSP1
and TSP2 are additive, Agah et al. [22] examined the
wound-healing response in double TSP1/TSP2-null mice.
Surprisingly, the phenotype of the response in double-null
mice resembled that of the TSP1-null mice and lacked
many of the features seen in TSP2-null mice, such as
increased angiogenesis and accelerated healing. This
unexpected finding was attributed to the fact that TSP1 is
expressed before TSP2 in the wound-healing response.
TSP1 is a potent chemotactic factor for inflammatory cells
[25]; in its absence the recruitment of neutrophils and
monocytes/macrophages that initiate the inflammatory
phase of wound healing is delayed and reduced. Thus, the
lack of TSP1 dictates the course of wound healing in
double TSP1/TSP2-null mice. These findings serve to
raise an important cautionary note at a time when test tube
experiments with purified reagents are commonly used to
establish the function of a protein. Whereas the intrinsic
properties of TSP1 and TSP2 are similar, their physiological
roles are very different and undoubtedly reflect their distinct
temporal and spatial patterns of expression in the organism.
As mentioned above, matricellular proteins have been
shown to be de-adhesive in vitro. An interesting example
of de-adhesive activity in vivo is an inhibition of
chordomesodermal cell migration during Xenopus laevis
gastrulation by a Ca2+-binding sequence of SPARC [26]. In
fact, several of the phenotypes described in worms, frogs
and mice that are associated with overexpression or ablation
of SPARC could reflect impaired migration patterns of
specific cells during development [27]. Rempel et al. [28]
have demonstrated that clones of transfected glioma cells
expressing different levels of SPARC exhibited enhanced
or decreased rates of attachment and migration as a function
of the ECM substrate. Accelerated migration in vitro was
observed in dermal fibroblasts derived from SPARC-null

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Cell-to-cell contact and extracellular matrix

Table 2
Disruption of matricellular genes produces diverse and unexpected phenotypes.*
Gene

Phenotype

Refs

Thrombospondin-1

Epithelial and smooth muscle cell hyperplasia and inflammation in the lung; Evidence for less active
TGF-
1

[52,67]

Thrombospondin-2

Increased angiogenesis; bleeding diathesis; abnormal collagen fibrils; reduced fibroblast adhesion;
increased fragility of skin

[7,20,23,
33]

SPARC

Cataracts; accelerated wound healing; increased adipogenesis; increased fibrovascular response to

injury; osteopenia; immature collagen fibrils

[8,27
,29,
30,35]

Osteopontin

Reduced T-cell-mediated immunity; increased dystrophic calcification; reduced host response to injury;
reduced bone resorption

[9,10,68]

Tenascin-C

Subtle neurological/behavioral defects; suppression of hematopoietic activity; abnormal response to

injury

[11,69]

Tenascin-X

Reduced collagen content and fibril density in skin; increased tumor invasion and metastasis; increased
MMP2 and MMP9

[6,70]

In contrast to these phenotypes, mice that lack CTGF die shortly after birth (KM Lyons, personal communication).

mice. These animals not only exhibit more rapid closure of

excisional wounds, but also showed enhanced fibrovascular
invasion of subcutaneously implanted sponges [29,30].
Data from experiments in vivo are therefore consistent
with an inhibitory role of SPARC on cellular migration.
Initially, the many effects attributed to the activities of
matricellular proteins on cells might appear unrelated. In
Figure 1 we have attempted to summarize data from a
number of laboratories and to propose a common theme:
matricellular proteins function primarily to induce an intermediate state of adhesion through their effects on ECM,
growth factors and MMPs. This adhesive state affects
downstream signaling mediated by integrins, or other
ECM receptors such as syndecans and growth factor receptors,
with several possible outcomes. Intercommunication of
integrins and growth factor receptors is critical for the
establishment of signaling networks within the cell.
Efficient integrin engagement with the ECM has been
shown to result in growth arrest (as with 21 and fibrillar
collagen) or stimulation (e.g. 21 and monomeric collagen,
or 51 and fibronectin) [31]. In some instances, unligated
integrins, or expression of integrins in the absence of
ligand, can lead to cell death [32].

Matrix assembly and collagen fibrillogenesis

A prominent feature of the TSP2-null mouse is fragility of
skin and laxity of tendons and ligaments (Table 2). Dermal
collagen fibers are abnormal by light microscopy, and
fibrils in both skin and tendons are larger in diameter and
irregularly contoured [33]. Although TSP2 could not be
detected as a collagen-fiber-associated protein by immunocytochemistry of uninjured skin, an association of the
protein with collagen fibers was observed under conditions
of increased synthesis, for example as occurs in a healing
excisional wound [7]. We do not understand the mechanism
by which TSP2 modulates fibril size, but certain clues

exist. For example, examination of early postnatal tendons

from TSP2-null mice revealed that the compartmentalization of the ECM by the cytoplasmic processes of mutant
tendon fibroblasts was disrupted, and the processes themselves were disorganized. Furthermore, the apposition of
growing collagen fibrils to the cell surface was less close
than was seen in control cells [34]. While not diagnostic,
these changes could result from increased proteolytic
activity at the cell surface of TSP2-null cells, for example
by MMP2.
Unlike findings in TSP2-null mice and patients with some
forms of the EhlersDanlos syndrome, tenascin-X deficiency
in mice and humans does not lead to defects in collagen
fibrillogenesis [6]. However, the collagen content of skin
in tenascin-X mice is reduced, and collagen fibril density is
decreased. How tenascin-X regulates collagen matrix
assembly is not understood. Although OPN is found in
bone in the absence of growth or injury, this protein does
not appear to contribute to the structural integrity of the
tissue, since bone in the uninjured OPN-null mouse is normal.
The primary functions proposed for OPN in calcified
tissues are regulation of bone-cell adhesion and osteoclast
function. In soft tissues and in fluids such as urine and
milk, OPN has been implicated in the inhibition of
mineralization in response to injury and in the formation of
calcium crystals [10].
The different abnormalities noted in mice with a targeted
disruption of the SPARC gene (Table 2) might reflect a
general defect in the production or assembly, or both, of
ECM and connective tissue. Consistent with the abundance
of SPARC in normal bone and its reduction in several
diseases characterized by osteopenia, SPARC-null mice
exhibited significantly decreased bone formation, attenuated
levels of osteoblasts and osteoclasts, and impaired remodeling
[35]. The interaction of SPARC with collagen-I, its regulation

Matricellular proteins: extracellular modulators of cell function Bornstein and Sage

611

Figure 1
Matricellular proteins affect receptors that
mediate both adhesion and proliferation. This
hypothetical scheme is consistent with data
describing the regulation of cell behavior and
cellECM interactions by several of the
matricellular proteins (see text). Matricellular
proteins induce an intermediate state of
adhesion in anchorage-dependent cells
through their regulatory effects on ECM
production and assembly, and growth factor
and MMP activity. This adhesive state results
in signaling by both integrins and growth
factor receptors (GF-Rs) Stimulation of
growth is effected by efficient ligation of
integrins and activation of GF-Rs, whereas
growth arrest occurs in cells in which
integrins are inefficiently ligated. In the
extreme case, unligated integrins can lead to
apoptosis. In addition, matricellular proteins
can influence cell behavior by interactions
with other cell-surface receptors.
P, phosphorylated protein; R, receptor.

MATRICELLULAR PROTEINS

ECM

Alterations in:
GF activity

MMPs

Intermediate state of adhesion

INTEGRINS

GF-Rs

Efficient ligation

GROWTH
STIMULATION

R-P and downstream

signaling

Inefficient binding,
unoccupied receptors

GROWTH
ARREST

Inhibition of
R-P and MAPK-P

Compromised
interactions with ECM

APOPTOSIS

Abrogation of
survival signals
Current Opinion in Cell Biology

of angiogenesis, and its modulation of MMP and growth

factor activity are possible causative factors contributing to
the phenotype in bone.
Three other intriguing abnormalities have been described
in SPARC-null mice: cataracts, enhanced adipose tissue,
and lax skin. Is there a common theme among these seemingly disparate characteristics? Dermal collagen fibrils in
SPARC-null animals are smaller and more uniform in size
[27,29]. These differences were observed in several models
of dermal injury and are consistent with the reduced collagen
output of certain SPARC-null cells [36]. The dermis and
abdominal cavity of SPARC-null mice also contained
excessive deposits of fat (AD Bradshaw, personal communication). Interestingly, the accumulation of fat was not
accompanied by an increase in body weight, an observation
indicating compensatory loss of other tissue mass (e.g. bone).
An explanation for the aberrant adipogenesis in SPARCnull mice was suggested by studies of the cataracts in these
animals [27]. Yan et al. [37] found that the lens capsule
basement membrane is defective in mice lacking SPARC.
In addition to the frequent extensions of lens epithelial
cell processes into the capsule, both collagen-IV and
laminin-1 were deposited abnormally in this basement
membrane. A consequence of altered synthesis and/or
assembly of the major structural basal lamina components
is an enhanced permeability to water and small molecules
that appears to be linked to the swollen lens fibers and
cortical cataracts that occur shortly after birth. Since
mature adipocytes are each surrounded by a basal lamina,

and, like lens epithelial cells, rely on ECM and growth-factormediated signaling for differentiation, it is possible that
their altered size and proliferation result from an impaired
ability to produce and assemble collagen-IV or laminin-1
into a functionally competent basement membrane.

Regulation of proliferation and apoptosis

Most matricellular proteins appear to affect cell proliferation.
Although it is highly probable that regulation of the cell
cycle and modulation of cell adhesion are related responses
to at least some matricellular proteins, precise mechanisms
remain to be identified. It is now appreciated that signals
from both integrins and growth factor receptors are necessary for G1 S-phase transition and cell proliferation [31].
Synergy between these receptors results from input generated
by the binding of ECM ligands and growth factors that act
on different components of the same signaling pathways
(Figure 1). Engagement of integrins can trigger the activation
of, for example, mitogen-activated protein kinases
(MAPKs), Rac and the JAKSTAT pathways, whereas
growth factor receptor tyrosine kinases, activated by the
plating of cells on selected ECM proteins, can stimulate
integrins to recognize different extracellular ligands [38].
In light of the important role of matricellular proteins in
cell adhesion, it is perhaps not surprising that these proteins
also influence the cell cycle.
SPARC added to cultured cells inhibits their proliferation
[4,8]. Predictably, a wide range of cell types cultured from
SPARC-null mice exhibit markedly accelerated cell cycles,

612

Cell-to-cell contact and extracellular matrix

which can be rescued by the addition of SPARC [39]. A

minimum of two signaling pathways were identified that
mediated the de-adhesive and anti-proliferative effects
of SPARC on cells, the former being tyrosine-kinasedependent, and the latter relying, in part, on heterotrimeric
G-protein-coupled signal transduction [8]. Divergence of
these two pathways could offer a partial explanation for
the resistance of transformed cells to SPARC-induced
de-adhesion, whereas the G1S-phase transition of nearly
all cells is inhibited to some degree by SPARC.
In a recent study [40], smooth muscle cells (SMCs), plated
on a thin film of collagen without exogenous growth factors,
were growth-arrested by SPARC in late-G1 in the absence
of effects on cell shape or on the levels of cyclin-dependent kinase (cdk) inhibitors [40]. The effects of SPARC
were associated with reductions in the phosphorylation of
the retinoblastoma protein (pRb), in the levels of cyclin A
and the pRb-related protein p107, and in the diminished
activity of cyclin E-associated cdk-2. Thus, in addition to
its direct interaction with platelet-derived growth factor
(PDGF), a major growth factor for these cells, SPARC
antagonizes their cell cycle progression at the level of cdk-2
activity. This latter effect might be especially important
when cells are subjected to injury and breakdown of ECM,
whereas the former would be more relevant during cellular
quiescence in an established ECM.
SPARC also induces apoptosis, specifically in human ovarian
carcinoma cells [41]. Although a molecular pathway
directing this activity has not been identified, SPARCmediated cell killing indeed appears to control malignancy
in ovarian epithelium. In contrast, OPN appears to deliver
anti-apoptotic (survival) signals to cultured cells, although
the molecular targets appear to be cell type-specific [42].
For example, survival in endothelial cells (ECs) is
dependent on the activation of NF-B, whereas survival in
cells dependent on signaling via the common subunit of
interleukin-3 or granulocytemacrophage colony-stimulating
factor involves CD44 and its activation of the phosphatidylinositol 3-kinase/Akt cascade [42,43]. In the latter
case, cooperation between OPN-bound CD44 and a
growth factor receptor establishes a positive autoregulatory
loop that inhibits apoptosis.
The effects of TSPs on cell proliferation are also complex
and dependent on cell type. TSP1 acts synergistically with
growth factors to stimulate the proliferation of SMCs and
also promotes the clonal expansion of T cells [12]. On the
other hand, the ability of TSP1 to serve as an inhibitor of
angiogenesis is supported by many experimental assays,
in vitro and in vivo [12,13,14,44,45]. For example, TSP1
inhibits the migration and proliferation of ECs and their
ability to form tubes. TSP1 also inhibits neovascularization
in response to basic fibroblast growth factor (FGF-2) in
rodent corneas and to vascular endothelial growth factor
(VEGF) in chick chorioallantoic membranes (CAMs), and
in subcutaneously implanted sponges. The inhibition of

angiogenesis by TSP1 involves engagement of the scavenger

receptor CD36, and activation of pp59fyn, and the stressand mitogen-activated kinases, JNK and p38. These
events lead to activation of caspase 3 and induction of
apoptosis [46,47]. Specificity for remodeling vessels is
provided by the dependence of TSP1-induced apoptosis
on FasFas ligand interactions, which occur preferentially
in capillary sprouts [48].
Induction of caspases and cell death may not be the only
way in which TSPs inhibit angiogenesis, however. We
have found that VEGF-stimulated proliferation of human
microvascular ECs was inhibited by either recombinant
TSP1 or TSP2, without a significant increase in caspase
activity or evidence for apoptosis [49]. This inhibition was
accompanied by a marked decrease in the percentage of
cells in the G2/M and S phases of the cell cycle. We note
that many of the studies that implicate apoptosis of ECs as
the basis for the inhibition of angiogenesis by TSPs were
performed with cells plated on gelatin in the presence of
low serum, or with corneal assays that also utilize a collagen
substratum in a plasma-protein-poor environment. These
conditions could be conducive to apoptosis (Figure 1). The
apparently conflicting results could reflect the propensity
for TSPs to interact with different cell-surface receptors in
a context-dependent manner. Thus, both mechanisms
could function in inhibition of angiogenesis.

Binding/activation of growth factors and

cytokines; regulation of growth factor production
SPARC has been shown to abrogate cellular responses to
PDGF, VEGF and FGF-2, although the mechanisms vary
and are incompletely defined [8]. In addition to its highaffinity binding to PDGF-B and VEGF, SPARC was shown
to compete for the binding of PDGF to fibroblasts, and
to interact specifically and saturably with SMC via its
carboxy-terminal domain [8,40]. Consequences of the
binding of SPARC to VEGF165 include prevention of
VEGFVEGFR1 (VEGF receptor 1) interaction, inhibition
of VEGFR1 phosphorylation, and diminution of MAPK
(ERKs 1 and 2) phosphorylation, which collectively result
in an inhibition of human microvascular EC proliferation
by >90% [8]. A somewhat different mechanism accounts
for the inhibition of FGF-2-mediated effects on cell division
and differentiation. In the absence of a direct interaction
with FGF-2, SPARC prevented the phosphorylation of
FGFR1 and enhanced the differentiation of MM14
myoblasts into myocytes [8]. Thus, the expression of
SPARC could reduce the levels of certain growth factors
that might otherwise stimulate excessive cellular proliferation.
Alternatively, SPARC could minimize growth factor cognate receptor activity at the cell surface by its interaction
with appropriate co-receptors to prevent receptor dimerization,
or with other effectors of growth factor signal transduction.
Culture of SPARC-null mesangial cells revealed a reduction
of collagen-I mRNA and protein that was attributed to the
significantly diminished synthesis of transforming growth

Matricellular proteins: extracellular modulators of cell function Bornstein and Sage

factor 1 (TGF-1) by these cells [36]. Although SPARC

does not appear to activate latent TGF-1 directly,
SPARC-null mesangial cells show depressed levels of
phosphorylation of the TGF-1-dependent signal transduction
protein, Smad 2 [50], as well as the transcription factor
ATF-2 [8]. The persistent alterations in ECM seen in
SPARC-null mice are in all probability linked to a diminished
production of TGF-1 in some tissues, a deficit that could
influence fibrosis and other responses to injury.
The ability of TSP1 to activate latent TGF-1 has
received widespread attention and is well documented by
experiments in vitro [51]. However, the evidence for a
physiological role of TSP1 in the generation of active
TGF-1 in vivo is conflicting. Support for such a role is
provided by the findings that there are similarities in the
histopathological changes between TSP1- and TGF-1null mice, and that these changes can be reversed by
treatment of postnatal TSP1-null pups with an activating
peptide, KRFK, from TSP1 [52]. Active TGF-1 levels
were also increased in extracts of melanoma tissue from
mice treated with a recombinant fragment of TSP1 [53].
On the other hand, the levels of active TGF-1 were not
reduced in platelets from TSP1-null mice [54], and the
ratio of active to total TGF-1 was normal in healing excisional wounds from TSP1- and TSP1/TSP2 double-null
mice [22]. In the latter case, levels of total TGF-1 were
significantly reduced, possibly because TSP1 normally
serves as a potent chemoattractant for TGF-1-producing
monocytes/macrophages. Thus, while it is evident that
activation of latent TGF1 by proteases and other protein
interactions can compensate for a lack of TSP1, it is also
possible that some of the changes in TSP1-null mice that
have been attributed directly to reduced activation of
latent TGF1 could result from abnormalities in recruitment
of inflammatory cells.

Angiogenesis and tumor growth

It is now generally accepted that most primary and
metastatic neoplasms require an angiogenic response on
the part of the host for their growth [55]. As inhibitors of
EC proliferation, TSP1 and TSP2 were prime candidates
for studies of their efficacy as endogenous inhibitors of
tumor angiogenesis. The incidence of tumors in mice bearing
the neu oncogene under the control of the MMTV promoter
was increased after these mice were placed on a TSP1-null
background, and was reduced by co-expression of an
MMTV-hTSP1 transgene [44]. In these studies TSP1 was
shown to inhibit the activation of pro-MMP9 by MMP3,
consistent with its anti-angiogenic activity, and with its
function as a direct-binding inhibitor of several other
enzymes [56]. Tumors in nude mice produced by A431
squamous carcinoma cells, stably transfected with either
TSP1 or TSP2 cDNAs, were smaller and less highly
vascularized than controls, and cells transfected with both
cDNAs produced no palpable tumors [57]. It is of interest
that TSP2 was considerably more effective in the inhibition
of tumorigenesis in this model than was TSP1. TSP2

613

mRNA was strongly upregulated in the stroma of chemically

induced skin carcinomas in control mice, in contrast to the
reduced expression of TSP1, and TSP2-null mice were
more susceptible to the induction of tumors [58]. The
resulting tumors in TSP2-null mice grew more rapidly,
were more vascular, and contained fewer apoptotic cells,
relative to the controls. Paradoxically, in view of the
evidence for the anti-angiogenic activity of TSPs in animals,
the amino-terminal fragment of TSP1 is capable of stimulating
EC proliferation and angiogenesis in a corneal or CAM
assay [59,60].
There are numerous points during the growth, regression
and remodeling of vessels at which SPARC and other
matricellular proteins could exert regulatory effects. An
overall inhibitory function for SPARC in vascular growth is
indicated by the enhanced fibrovascular invasion of
sponges in SPARC-null mice [30]. Interpretation of this
result is complicated by the observation that SPARC, as
well as OPN, several collagens, and other proteins, can
be cleaved proteolytically into fragments with angiogenic
activities different from those of the parent protein
[6163]. Copper-binding peptides that can be released
from the follistatin domain of SPARC by plasmin or
MMPs, and which contain the sequence GHK, stimulate
EC proliferation, angiogenesis in CAMs, and ECM
production during wound healing [8,64]. Thus SPARC can
be angio-stimulatory as well as inhibitory.
Matricellular proteins also feature importantly in the
regulation of tumor-cell behavior. Descriptive data abound
here, as most of these proteins are expressed at high levels,
either by the tumor cells themselves or by other cells in
their vicinity. Production of SPARC is characteristic of
invasive gliomas, meningiomas and melanomas, and inhibition
of SPARC in melanoma cells by anti-sense RNA blocked
tumorigenicity in vivo [8]. A potential mechanism for the
latter result might lie in the induction by SPARC of an
intermediate state of adhesion that is permissive for migration
on vicinal ECM. Alternatively, the regulation of MMPs by
SPARC could be a major factor in tumor-cell growth and
invasion. In invasive breast and prostate cell lines, SPARC
was shown to increase the levels and activity of MMP-2,
which degrades ECM and affects vascular growth, in part
through its association with the integrin v3 [8]. The
ability of SPARC to enhance endothelial permeability
would increase vascular leak and the migration of tumor
cells into the bloodstream. Resolution of these proposed
functions is now feasible with tumor models in SPARCnull mice.
It is of interest that all six CCN family members contain a
type I TSP repeat, but the two best characterized, CYR61
and CTGF, are pro-adhesive and angiogenic [5,65]. The
ability of CYR61 to promote cell adhesion and migration,
and enhance growth-factor-stimulated EC proliferation is
consistent with its angiogenic activity. However, CTGF
has also been shown to bind VEGF, inhibit the binding of

614

Cell-to-cell contact and extracellular matrix

the latter to VEGFR2, and inhibit VEGF165-induced

angiogenesis in a subcutaneously implanted matrigel plug
[66]. Thus, like matricellular proteins, the functions of CCN
proteins may be contextual and depend on the identity of
the growth factors that predominate in the local environment.

Conclusions and future directions

The information that cells receive from their environment
is provided by a variety of molecules that range from
growth factors and cytokines to adhesive proteins and
proteoglycans. Until recently, little attention was paid to
mechanisms that might coordinate or modulate this flow of
information, either at the level of cell-surface receptors or
at a distance from the cell. We suggest that matricellular
proteins perform this regulatory function in different ways,
some of which have been discerned and are described
in this review. Thus, matricellular proteins are capable of
sequestering growth factors (e.g. VEGF or PDGF and
SPARC), binding ions (e.g. Ca2+ and OPN), inhibiting
proteases by direct binding (e.g. serine proteases or MMP3
and TSP1), clearance of proteases by endocytosis (e.g. MMP2,
LRP and TSP2), and activating cytokines (e.g. latent
TGF-1 and TSP1). Furthermore, some matricellular proteins,
such as TSP1 and TSP2, bind a bewildering number of
cell-surface receptors. The mapping of the pathways and
networks activated by these interactions represents a
daunting task for future research. Paradoxically, a signaling
receptor for SPARC has not yet been identified, but the
search continues.
Perhaps the most important lesson that the study of matricellular proteins has taught us is that the functions of these
proteins can only be fully appreciated within the context of
the whole organism. This conclusion is well illustrated by
the distinction that can be made between the functions of
TSP1 and TSP2 in wound healing, as revealed by analyses
of TSP1/TSP2 double-null mice [22], whereas numerous
experiments in vitro had indicated nearly equivalent
functions. Arguably, the role of SPARC in adipogenesis
and in the homeostasis of the lens [37] could only have
been gleaned from studies of SPARC-null mice. These
and other unexpected insights, notably the role of OPN in
T-cell-mediated immunity, suggest that continued study
of matricellular proteins is likely to be rewarded with
fascinating new findings.

Acknowledgements
We thank A Agah, L Armstrong, T Kyriakides, J Lawler and J MurphyUllrich, for helpful comments on the manuscript. Original studies from the
authors laboratories were supported by National Institutes of Health grants
AR 45418, HL 59475, EY 13180, GM 40711, and National Science
Foundation grant EEC 9529161.

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